Perinatal hypoxia-ischemia (HI)represents a long-standing, refractory public health problem. The neurologic sequelae of the severe form of this injury include mental retardation, epilepsy, cerebral palsy and blindness. Thus, the ability to identify therapies specific to the immature brain is of clinical importance. Recent data from our laboratory and others, indicate that the developing brain is more vulnerable to HI injury than the mature brain, due to a deficiency in anti-oxidant enzyme capacity. However, how this increased oxidative stress produces injury to the developing brain remains unresolved. Our recent data indicate that HI increases reactive oxygen species (ROS) generation in the neonatal brain. However, we have also found that increases in ROS are only associated with brain injury in the presence of an active nitric oxide(NO) synthase (NOS). Our data also indicate that perinatal HI leads to increased generation of the hydroxyl radical and that this is NO-dependent. Based on this data, in this competitive renewal, we will test the overall hypothesis that the injury associated with HI in the developing brain occurs through the convergence of both NO-dependent and NO-independent signaling pathways. We hypothesize that HI stimulates the activity of the NADPH oxidase complex, which in combination with the activity of superoxide dismutase generates hydrogen peroxide. Simultaneously, the activation of NOS leads to increases in NO. We hypothesize that NO modulates brain iron homeostasis leading to an increase in the levels of free iron. The reaction of the free iron with the increased hydrogen peroxide leads to the generation of hydroxyl radical. We hypothesize that the increase in hydroxyl radical, rather than superoxide or hydrogen peroxide, is the key mediator of neuronal loss after HI. We will test this overall hypothesis by determining the mechanisms by which: 1) HI activates the NADPH oxidase enzyme complex; and 2) Hydroxyl levels are increased by HI. It is anticipated that the data obtained will identify signaling intermediates that could be targeted to produce new therapeutic agents for infants and children exposed to asphxia.